Ethical approval
The Institutional Review and Animal-Care Boards of Ataturk University Faculty of Medicine approved the study. All the investigations and procedure were made in agreement with nationwide guiding principles on behalf of the practice and research animal care. The study was blinded. The conduct of experiments (DK, IDS, AK), outcome assessment (MY), and data analyses (BS, IC, FA) were independently done.
Experimental design
Forty-eight female Wistar albinos were enrolled those were gained from our Research Laboratory. They were 3–4 months old, 250–300 g, and healthy and never took part in any other experiment before. The groups had been created as shown in Fig. 1 at the stated conditions. The cages (classic plastic confine on sawdust bedding), temperature (22 °C), light (14/10 h light/dark cycle), and feeding (standard rat food and tap water ad libitum) were controlled and uniformity was ensured.
A 1-day water withdrawal and intramuscular injection of 25% glycerol (Bikar Medical Products, Istanbul, Turkey) prompted renal damage in groups of II, IV, VI, and VIII with a dosage of 10 ml/kg to gluteal muscle. Twenty-four hours after glycerol injection, intravenous delivery of 10 ml/kg iohexol (Omnipaque, Opakim Medical Products, Istanbul, Turkey) via tail vein stimulated nephrotoxicity. Thirty minutes after glycerol injection, levosimendan was injected intraperitoneally to the V, VI, VII, and VIII groups. Levosimendan administration was continued for the second, third, and the fourth days.
The experiment was completed on the 5th day of glycerol injection by the high dose of thiopental sodium (50mg/kg). Under general anaesthesia, blood specimens were taken from the cardiac cavity. Kidneys were excised straightaway for biochemical evaluations, molecular analyses, and pathological examination.
Blood urea nitrogen, creatinine, and biochemical analyses
Serum blood urea nitrogen (BUN) (Lot number B0382A, S.r.i, Italy) and creatinine (Lot number B0914A, S.r.i, Italy) were identified using commercially existing supplies. Analyses were done in a ChemWell 2910-automated-EIA and chemistry analyser (Awareness Technology, Inc., Palm City, FL, USA).
After surgical excision, the kidneys were stored at −80 °C. The tissues were initially perfused by phosphate-buffered saline (PBS)/heparin and then ground in liquid nitrogen using the TissueLyser II grinding jar set (Qiagen, Hilden, Germany). Approximately 100 mg of ground tissue was homogenised in 1 mL PBS homogenate buffer in an Eppendorf tube with TissueLyser II, and the samples were then centrifuged. Superoxide dismutase (SOD) activity, glutathione (GSH), and malondialdehyde (MDA) levels from supernatants and standards were measured at room temperature in duplicate via modified methods, with an enzyme-linked immunosorbent assay, ELISA, reader. A standard curve was plotted and the equation was obtained from the absorbance of the standards. The linear SOD, GSH, and MDA concentrations were calculated according to this equation and were expressed as U/mg-protein, nmol/mg-protein, and nmol/mg-protein, respectively. The data obtained were presented as mean ± standard deviation as 1-mg protein.
Molecular analyses
Total ribonucleic acid (RNA) extraction and complementary deoxyribonucleic acid (cDNA) synthesis
The tissues (20 mg) were stabilised in RNA stabilisation reagent (RNAlater, Qiagen, Hilden, Germany), and then disrupted using the TissueLyser II (Qiagen, Hilden, Germany). Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) in a QIAcube (Qiagen, Hilden, Germany), based on the manufacturer’s instructions. The RNA samples were then reverse-transcribed into cDNA: using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). From 10 μL, total RNA was treated with 2 μL 10X RT buffer, 0.8 μL 25X dNTP mix, 2 μL 10X RT random primers, 1 μL multiscribe reverse transcriptase, and 4.2 μL DEPC-H2O. Reverse transcription was conducted at 25 °C for 10 min, followed by 120 min at 37 °C, and finally, 5 min at 85 °C, using a Veriti 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA). The concentration and quality of the cDNA were assessed and quantified using the Epoch Spectrophotometer System and Take3 Plate (BioTek Inc., Winooski, VT, USA).
Relative quantification of gene expression
Relative tumour necrosis factor-α (TNF-α), nuclear factor kappa beta (NFK-ß), and interleukin-6 (IL-6) messenger RNA (mRNA) expression analyses were performed using StepOne Plus Real-Time polymerase chain reaction (PCR) system technology (Applied Biosystems, Foster City, CA, USA) using synthesised cDNA from rat kidney RNA. A quantitative PCR was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using primers generated for rat TNF-α Rn00562055_m1, rat NFK-ß Rn01399583_m1, rat IL-6 Rn01410330_m1, and rat β-actin Rn00667869_m1. The results are expressed as relative-fold, compared with control animals. The expression data for β-actin in each tissue were used as the endogenous control. Each determination was performed in triplicate for each tissue in a 96-well optical plate for both targets, using 9-μL cDNA (100 ng), 1 μL of Primer Perfect Probe mix, and 10 μL of QuantiTect Probe PCR Master Mix (Qiagen) in each 20-μL reaction. The plates were heated for 2 min at 50 °C and then 10 min at 95 °C. Subsequently, 40 cycles of 15 s at 94 °C and cycles of 60 s at 60 °C were conducted. All data are expressed as the fold-change in expression compared with the expression in other animal groups, using the 2-delta-delta Ct (2-ΔΔCt) method [13, 14].
Pathologic analyses
Kidneys of the rats in all groups were obtained, sectioned in coronal plane, and fixed at 10% neutral formalin for 48–72 h. The tissues were then routinely processed, and embedded in paraffin wax, and 4–5-μm thick serial sections were cut. All tissue sections were stained with haematoxylin and eosin for histopathological assessment and examined under a light microscope (Olympus BX51, Tokyo, Japan). Hyaline and haemorrhagic casts and tubular necrosis were evaluated and counted. A minimum of five fields for each kidney slide at 100× magnification was evaluated. The severity of the changes was evaluated using scores on the following scale: grade 0, negative; grade +1, mild; grade +2, moderate; grade +3, severe; and grade +4, most severe [14].
Statistical analysis
Statistical analysis was performed using SPSS 20.0 software (IBM Corp. SPSS Statistics for Windows, Armonk, NY, USA). Continuous variables are displayed as the mean ± standard deviation, and categorical variables are reported as counts and percentages. Comparisons of biochemical evaluations and molecular analyses among groups were performed using a one-way analysis of variance and Duncan’s multiple comparison tests. A p-value under 0.05 was set as statistically significant.